Immunohistochemical kit

Immunostaining for interleukin 6 (IL-6) was performed on 4 μm decalcified distal femoral sections. The procedures were performed as described previously7 (link) with an immunohistochemical kit (Boster, Wuhan, People’s Republic of China) by following the manufacturer’s instruction. The anti-IL-6 antibody was purchased from Abcam. The region of interest was the distal femur growth plate and the distal femur metaphysis located between 0 and 2 mm distal to the growth plate epiphyseal junction. The images in the region of interest of each sample were captured at 20× magnification. Six different images were randomly collected per sample and subjected to final analysis. The analysis was performed by the biomedical image analysis software (Image Pro plus 6.0, IPP 6.0; Media Cybernetics Inc., Silver Spring, MD, USA). The evaluation of IL-6 expression was determined by integral optical density values of the images. The integral optical density values of positive staining in the distal femur are presented as mean ± standard deviation. All image groupings analyzed using IPP 6.0 were blinded to the analysts.

Pure AST is provided by Shanghai Yuanye Biotechnology Co., Ltd. In the experiment, pure AST was dissolved with phosphate buffer (PBS) and diluted with saline. Pentobarbital sodium, papain, and cysteine were purchased from Guangzhou Chemical Reagent Factory. Hematoxylin, water-soluble eosin Y, toluidine blue O, paraformaldehyde, and Ethylene Diamine Tetraacetic Acid (EDTA) were from TAKARA, Japan. Anti- PEG2, β-actin, and other monoclonal antibodies were purchased from Abcam, UK. Finally, the immunohistochemical kit was purchased from Boster, Wuhan, China) The fluorescence quantitative PCR detection kit was purchased from Baoshi, Dalian, China, and the Prime Script RT kit were purchased from Takara, Dalian, China.

The brain tissues were fixed with 4% formaldehyde and cut into 5 mm-thick serial sections, and immunohistochemistry staining was performed according to the immunohistochemical kit (Boster Biological Technology, Wuhan, China) by using the specific cleaved caspase-3 antibody (1:300; ab2302, Abcam). A blinded investigator used a microscope to take images and chose images randomly for each section. The expression of cleaved caspase-3 was analyzed by the Image-Pro Plus software.